Concept

To make our DNA Screw, we started creating a Cylinder and a Ring made of DNA Origami. First, we made a cylinder and a ring separately. Every structure was observed by Atomic Force Microscope; AFM. We succeeded in making the Cylinder; however the image of the Ring did not represent the designed structure. Second, we made modified structure of the Cylinder and the Ring. The structure was made from a single scaffold strand, and the Cylinder and the Ring were connected, since the electrostatic interaction between the Cylinder and the Ring could be avoided. We could observe the structure by AFM. Finally, we synthesized the spiders and attached them to the cylinder; however we could not observe expected image by AFM.

Reagents

TAE buffer

Tris-acetate-EDTA buffer, which is essential to make DNA Origami.

Mg2+

Has a function to shape DNA Origami.

TE buffer

Has a function to preserve DNA.

TBE buffer

Tris-Borate-EDTA buffer, which is essential when making DNA tile and doing electrophoresis.

M13mp18

Scaffold strand which we bought from TAKARA BIO INC.

Results

We succeeded in making the Cylinder and the Cylinder-Ring and synthesizing DNA Spiders!!!

Methods

Cylinder(1st ver.)

We made a Cyliner by DNA Origami.

First, we made staple mix for the Cylinder. We put 2.5μL for each staple whose stock is 50μM into the tube and diluted to 250nM with TE buffer. We had a set of 179 staples and we had total amount of 500μL. After putting all the protocols into the tube, we vortexed for a few seconds.

Second, we made 100μL solution of the Cylinder. We made 10-equivalent and 15-equivalent. As for 10-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 6.25μL of M13mp18 (stock concentration: 40nM), 10μL of Staple Mix (stock concentration: 250nM), and 72.5μL of mQ into the tube. As for 15-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 6.25μL of M13 (stock concentration: 40nM), 15μL of Staple mix (stock concentration: 250nM), and 67.5μL of mQ. After putting all the protocols into the tube, we vortexed for a few seconds.

Third, we put the tube into an thermal cycler. We annealed it from 90°C to 20°C -0.2°C/min.

Here, we put the table of amount, final concentration and stock concentration of each protocols.

Staple Mix

Amount

Stock

Staple

2.5μL(each)

50μM

TE buffer

52.5μL

Total

500μL

10-equivalent

Concentration

Amount

Stock

Mg2+

12.5mM

1.25μL

1M

TAE10x

10μL

M13

2.5nM

6.25μL

40nM

Staple Mix

25nM

10μL

250nM

mQ

72.5μL

Total

100μL

15-equivalent

Concentration

Amount

Stock

Mg2+

12.5mM

1.25μL

1M

TAE10x

10μL

M13

2.5nM

6.25μL

40nM

Staple Mix

37.5nM

15μL

250nM

mQ

67.5μL

Total

100μL

Ring (1st ver.)

We made a Ring by DNA Origami.

First, we made staple mix for the Ring. We put 5μL for each staple whose stock is 50μM into the tube and diluted to 500nM with TE buffer. We had a set of 80 staples and we had total amount of 500μL. After putting all the protocols into the tube, we vortexed for a few seconds.

Second, we made 100μL solution of the Ring. We made 10-equivalent and 15-equivalent. As for 10-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 12.5μL of M13mp18 (stock concentration: 40nM), 10μL of Staple Mix (stock concentration: 500nM), and 66.25μL of mQ into the tube. As for 15-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 12.5μL of M13 (stock concentration: 40nM), 15μL of Staple mix (stock concentration: 250nM), and 61.25μL of mQ. After putting all the protocols into the tube, we vortexed for a few seconds.

Third, we put the tube into an annealing machine. We annealed it from 90°C to 20°C -0.2°C/min.

Here, we put the table of amount, final concentration and stock concentration of each protocols.

Staple Mix

Amount

Stock

Staple

5μL(each)

50μM

TE buffer

100μL

Total

500μL

10-equivalent

Concentration

Amount

Stock

Mg2+

12.5mM

1.25μL

1M

TAE10x

10μL

M13

5nM

12.5μL

40nM

Staple Mix

50nM

10μL

500nM

mQ

66.25μL

Total

100μL

15-equivalent

Concentration

Amount

Stock

Mg2+

12.5mM

1.25μL

1M

TAE10x

10μL

M13

5nM

12.5μL

40nM

Staple Mix

75nM

15μL

500nM

mQ

61.25μL

Total

100μL

Agarose Gel Electrophoresis(Cylinder(1st ver.) and Ring(1st ver.))

We also tried Gel Electrophoresis to see whether we had only one structure.

First, we prepared 30 mL of gel. We put 0.525 % of Agalose, 3 mL of TBE 10x, 375 μL of Mg2+ (stock concentration: 1M) into the tube and diluted with mQ. After adding all the protocols into the tube, we vortexed for a few seconds.

Second, we poured the gel into the frame.

Third, we put the frame into the refrigerator and left it for 2 hours.

Next, we mixed 10 μL of the following protocols and 2 μL of loading buffer for each protocol. The protocols were the solution of Cylinder (10-equivalient), Cylinder (15-equivalent), Ring (10-equivalent), Ring(10-equivalent), Staple mix of Cylinder and the Ring, and M13.

Next, we poured 400 μL of TBE buffer into a container and were subjected to electrophoresis at 50 V for one and a half hour at 4°C.

Here, we put the amount, final concetration, and stock concentration of the protocols we used for the electrophoresis.

Concentration

Amount

Agarose

1.75%(wt/V)

0.525g

TBE10x

1x

3mL

Mg2+

12.5mM

375μL

mQ

up to 30mL

Total

30mL

Unfortunately, the gel did not seperate as we had expected. The concentration of DNA was not high enough to be seperated.

Cylinder (1st ver.) Retry

There might have been artificial errors during the last experiment, so we decided to make the Cylinder again. We double checked every step so that we wouldn't make artificial errors.
We followed exactly the same steps we took in the last experiment, and we used the same amount and kind of protocol as the last experiment.

Ring (1st ver.) Retry

There might have been artificial errors during the last experiment, so we decided to make the Ring agin. We double checked evey step so that we wouldn't make artificial errors.
We followed exactly the same steps we took in the last experiment, and we used the same amount and kind of protocol as the last experiment.

Observing Cylinder (1st ver.) by AFM

Cylinder 15-equivalent

This time, we succeeded in observing the Cylinder!

The scan rate was 0.996 Hz.

Observing Ring (1st ver.) by AFM

Ring 10-equivalent

Unfortunately, the structure was not something we had expected.

The scan rate was 0.996 Hz.

Cylinder-Ring

We made Cylinder-Ring structure with DNA Origami.

First, we made staple mix for the Cylinder-Ring. We put 2μL for each staple whose stock is 50μM into the tube and diluted to 200nM with TE buffer. We had a set of 198 staples and we had total amount of 500μL. After putting all the protocols into the tube, we vortexed for a few seconds.

Second, we made 50μL solution of the Cylinder-Ring. We made 10-equivalent and 15-equivalent. As for 10-equivalent, we put 0.625μL of Mg2+(stock concentration: 1M), 5μL of TAE 10x, 6.25μL of M13mp18 (stock concentration: 40nM), 12.5μL of Staple Mix (stock concentration: 200nM), and 25.625μL of mQ into the tube. As for 15-equivalent, we put 0.625μL of Mg2+(stock concentration: 1M), 5μL of TAE 10x, 6.25μL of M13 (stock concentration: 40nM), 18.75μL of Staple mix (stock concentration: 200nM), and 19.375μL of mQ. After putting all the protocols into the tube, we vortexed for a few seconds.

Third, we put the tube into an annealing machine. We annealed it from 90°C to 20°C -0.1°C/min.

Here, we put the table of the amount, final concentration and stock concentration of protocols used in Cylinder-Ring structure.

Staple Mix

Concentration

Amount

Stock

Staple

200nM

2μL

50μM

TE buffer

104μL

Total

500μL

10-equivalent

Concentration

Amount

Stock

Mg2+

12.5mM

0.625μL

1M

TAE10x

5μL

M13

5nM

6.26μL

40nM

Staple Mix

50nM

12.5μL

200nM

mQ

25.625μL

Total

50μL

15-equivalent

Concentration

Amount

Stock

Mg2+

12.5mM

0.625μL

1M

TAE10x

5μL

M13

5nM

6.25μL

40nM

Staple Mix

75nM

18.75μL

200nM

mQ

19.375μL

Total

50μL

Observing Cylinder-Ring by AFM

cylinder-ring structure

We successfully observed the cylinder-ring structure. A few cylinders and some ring shaped stractures were seen.

The scan rate was 0.996 Hz.

Ring (2nd ver.)

We made a Ring by DNA Tile just in case.

First, we diluted the oligo to 100 μM.

Second, we made staple mix for the Ring (2nd ver.). We put 2μL for each staple whose stock is 100μM into the tube and diluted to 1μM with TE buffer. We had a set of 12 staples and we had total amount of 200μL. After putting all the protocols into the tube, we vortexed for a few seconds.

Second, we made 50μL solution of the Ring (2nd ver.). We made 10-equivalent and 15-equivalent. As for 10-equivalent, we put 6.25μL of Mg2+(stock concentration: 100mM), 5μL of TAE 10x, 6.25μL of M13mp18 (stock concentration: 40nM), 2.5μL of Staple Mix (stock concentration: 1μM), and 30μL of mQ into the tube. As for 15-equivalent, we put 6.25μL of Mg2+(stock concentration: 100mM), 5μL of TAE 10x, 6.25μL of M13 (stock concentration: 40nM), 3.75μL of Staple mix (stock concentration: 1μM), and 28.75μL of mQ. After putting all the protocols into the tube, we vortexed for a few seconds.

Third, we put the tube into an thermal cycler. We annealed it from 90°C to 20°C -0.1°C/min.

Here, we put the table of the amount, final concentration and stock concentration of protocols used in Ring (2nd ver.) structure.

Staple Mix

Concentration

Amount

Stock

Staple

1μM

2μL

100μM

TE buffer

176μL

Total

200μL

10-equivalent

Concentration

Amount

Stock

Mg2+

12.5mM

6.25μL

100mM

TBE 10x

5μL

M13

5nM

6.25μM

40nM

Staple Mix

50nM

2.5μL

1μL

mQ

30μL

Total

50μL

15-equivalent

Concentration

Amount

Stock

Mg2+

12.5mM

6.25μL

100mM

TBE 10x

5μL

M13

5nM

6.25μM

40nM

Staple Mix

75nM

3.75μL

1μL

mQ

28.75μL

Total

50μL

Observing Ring (2nd ver.) by AFM

Ring (2nd ver.)

The image was not what we had expected. The cause could could be that we did not need M13 and the annealing time was too short.

The scan rate was 0.996 Hz.

Ring (2nd ver.) Retry

We tried making the Ring (2nd ver.) again. This time, we changed our protocols and annealing time.

First, we made staple mix for the Ring (2nd ver.). We put 10μL for each staple whose stock is 100μM into the tube and diluted to 5μM with TE buffer. We had a set of 12 staples and we had total amount of 200μL. After putting all the protocols into the tube, we vortexed for a few seconds.

Second, we made 50μL solution of the Ring (2nd ver.). We put 0.7μL of Mg2+(stock concentration: 1M), 5μL of TBE 10x, 25μL of Staple Mix (stock concentration: 5μM), and 19.3μL of mQ into the tube. After putting all the protocols into the tube, we vortexed for a few seconds.

Third, we put the tube into an thermal cycler. We annealed it from 90°C to 20°C -0.02°C/min.

Here, we put the table of the amount, final concentration and stock concentration of protocols used in Ring (2nd ver.) structure.

Staple Mix

Concentration

Amount

Stock

Staple

5μM

10μL

100μM

TE buffer

80μL

Total

200μL

Ring (2nd ver.)

Concentration

Amount

Stock

Mg2+

14mM

0.7μL

1M

TBE 10x

5μL

Staple Mix

2.5μM

25μL

5μM

mQ

19.3μL

Total

50μL

Observing Ring (2nd ver.) by AFM

Ring (2nd ver.)

There were not any structures which had similar size of the designed ones.

The scan rate was 0.996 Hz.

Cylinder(first ver.) + Common Footings and DNA Spiders with Four Walking Legs

This part of our experiment aimed at seeing if the walking legs actually work. Here we used DNA spiders with four walking legs, which are different from the DNA spiders described in the Design. We planned to mix common footing,DNA spider, and the cylinder(1st ver.) after the cylinder was synthesized followed by being annealed and the DNA spider was synthesized. Zn2+ starts the DNA spider's cleaving motion. The interval of the footings on cylinder(1st ver.) is too wide for the DNA spider to cross so once all the four legs cleave the occupied footings that they attach, they will be detached from the cylinder. Therefore, taking "before" picture by AFM in which the DNA spiders are attached on the cylinders' surface and "after" picture in which the spiders disappear on the surface are considered to be a proof of the walking legs' function.

Synthesis of DNA spider

The spider was synthesized in the following protocol; 10μL Streptavidin, 11.36μL walking leg, 6.5μL TE, and 6μL NaCl

Spider

Amount

Stock

Streptavidin

10μL

1.89µM

walking legs

11.36μL

100μM

</tr>

NaCl

6μL

</tr>

TE buffer

6.5μL

Mixing of Cylinder(first ver.) + common footings and DNA spiders

4.0 μl of the solution of the cylinder(1st ver.) which had been annealed for three hours and 16.0 μl of the DNA spiders were mixed into one tube and left it for 30 minutes at room temperature(20~25℃). The solution of the cylinder(1st ver.) which had been annealed for 20 minutes also received the same operation.

AFM observation

Spiders

There were not any structures which had similar size of the designed ones.